This project will continue our investigation of the role of protein phosphorylation in regulating neuronal function. The biochemical mechanisms controlling neurite extension will be examined in NGF treated PC12 cells. Recent research in this laboratory has identified a novel proline-directed protein kinase activated by NGF treatment. The minimal recognition sequence for this kinase is --X-Ser/Thr-Pro-X--. Many neuron specific structural proteins including synapsin, tau, microtubule associated proteins, and neurofilament proteins contain this consensus sequence and several have been identified as in vitro substrates of this novel kinase. Since these proteins are substrates for more than one protein kinase and contain multiple phosphorylation sites, the specific sites of phosphorylation will be isolated and sequenced. The phosphorylation sites will be localized in the proteins that have a known sequence. These studies also will investigate the in situ phosphorylation of the cytoskeletal apparatus during neurite outgrowth. 32p-orthophosphate labelled PC12 cells will be treated with NGF and key cytoskeletal proteins will be examined for phosphate incorporation. Specific proteins including TH, synapsin, Tau, MAP's, and neurofilament proteins will be examined for their ability to incorporate phosphate. Since these same proteins are phosphorylated in situ following NGF treatment, the specific phosphorylation sites will be identified to examine the role of PDPK in mediating NGF induced neurite outgrowth. Other work in our laboratory has established that sphingosine, a specific inhibitor of PK C, blocks neurite extension. The exact role of PK C in modulating neurite extension will be further investigated in regard to specific substrates and sites phosphorylated during NGF induced neurite outgrowth. The relationship between PK C and PDPK mediated protein phosphorylation in regulating neurite extension will be examined. Understanding how neurons use protein phosphorylation mechanisms to form neurites may identify fundamental molecular mechanisms that underlie the pathogenesis of Alzheimer's disease, since the PDPK substrate proteins found in the neurofibrillary tangles have abnormal phosphorylation patterns.